Protein Expression

In my previous entry, I mentioned that we would move on to actually expressing our protein constructs. The procedure for protein expression was filled with its own issues – finding the ideal conditions to get the bacteria to express our plasmid has been very slow-going, and involved a lot of trial and error.

The major factors we have been altering in order to optimize protein expression are temperature and arabinose concentration. Arabinose acts as an activator for the expression of our protein; it allows the bacteria to express that gene. Increasing arabinose concentration should, theoretically, increase expression; however, it important not to add too much arabinose, because that could cause issues with expression. An additional problem is that TRα, in high concentrations, is toxic to bacterial cells. We want to find a level of expression that will give us enough protein to work with, but will also allow the bacterial cells to live long enough that they can produce enough protein.

Temperature can also affect which genes are being expressed, though how it would affect the expression of our plasmid is more difficult to say. We have tried increasing and decreasing the temperature from what was in the protocol, with mixed results.

The protocol involved growing bacteria in liquid culture, and then activating the expression of our plasmid by using arabinose, as described above. Samples of bacterial culture would be taken at hourly intervals, for four or five hours. Afterward, to get an idea of whether or not our protein was expressed, we would run a procedure called SDS-PAGE.

PAGE stands for Polyacrylimide Gel Electrophoresis. After lysing the cells, we would denature the proteins in the cell lysate by boiling them in a hot water bath and adding DTT; DTT also gives our protein a negative charge.  We could then load our samples onto the gel. An electric current would run through the gel, and the protein, being negatively charged, would flow through the gel toward the positive electrode. Larger proteins would have more trouble moving through the gel and would move less distance than smaller proteins, which could pass through the gel more easily. This way, SDS-PAGE allows us to separate out the protein mix by size, like taking things out of a messy drawer and separating them into categories.

Each size of protein will show up as a distinct band on the gel. We already had an idea of how large our fusion proteins should be; we knew the size of both the tags and each of the domains of TRα. If our proteins were expressed properly, we would see a large, dark, distinct band at the proper size of our protein, that would get darker and more distinct over time (indicating that expression increased).

We are still working on optimizing the expression of our protein. The process has been largely trial and error, changing one factor to see if there is any improvement. We have been getting some promising results, and we think we have a better idea of what conditions may be ideal.